The isolation of a specific subset of cells from a heterogeneous population of cells is necessary for a range of research and diagnostic tools. For example, isolation of circulating tumor cells (CTCs) from a buffy coat formed from a patient blood draw has shown clinical relevance. As is known, however, CTCs within the circulation of patients with metastatic cancer are very rare. More specifically, there is approximately one CTC per billion background cells. Further, the prognostically relevant bar for determining overall survival and disease-free progression of a patient is 5 CTCs per 7.5 milliliters (mLs) of whole blood. As such, CTC capture is an isolation method requiring both high sensitivity (5 cells) and high specificity (7.5 billion background cells). In addition, once captured, clinically relevant cellular analyses must be performed on the CTCs downstream of isolation.
While offering the flexibility to perform a wide range of downstream assays, macroscale methods to perform MC isolation have been found to be unsatisfactory. More specifically, macroscale methods to isolate these types of cells often require long, expensive and laborious procedures that may result in significant sample loss due to wasteful transfer steps or centrifugation and re-suspension steps, Capturing rare cells such as CTCs, which occur at frequencies on the order of 5-50 cells per 7.5 mL blood sample, is not feasible using traditional macroscale methods, as the loss of a single cell can represent up to a 20% loss of sample.
In order to overcome cell loss associated with the macroscale methods, heretofore described, microfluidic methods have arisen. Microfluidics offers novel solutions to the challenges of macroscale methods by providing a highly controlled, low-volume platform that can quickly and efficiently isolate cells. Further, microfluidic platforms offer sensitivity and specificity that is unattainable using current macroscale systems. Established microfluidic methods include functionalized micropost arrays, patterned surfaces and microfluidic systems that leverage density or other physical characteristics to isolate cells of interest from non-target cells.
In particular, the ability to use functionalized paramagnetic particles (PMPs) in microfluidic systems to isolate analyte of interest has expanded the utility of isolation methods across a range of platforms. One of PMPs advantages is that the particles are flexible for use in many system configurations since only a magnet is required for actuation and analyte isolation.
The ways to isolate an analyte of interest from a given sample can further divided into two basic methods. First, in the current primary method for using PMPs, the PMPs are held stationary while fluid is washed over the substrate to remove the background sample and any contaminants. Limitations of this popular method include the loss of the original input sample, allowing only a single effective isolation per sample, and the inefficiency of dilution-based sample preparation techniques, thereby necessitating multiple washes to effectively remove contaminants and leading to lengthy workflows. Second, recent work has demonstrated the ability to remove the PMPs from the original sample of interest using exclusion-based methods. These methods generally leverage gravitational forces or the dominance of surface tension at the microscale to position original samples and physically drag the PMPs out of the input sample along the surface of a device through some immiscible phase (e.g., air or oil) and into a second aqueous phase. These methods have been highly effective at isolating analyte with high specificity and selectivity. Further, these methods have been beneficial for their elegant workflow since isolation can be performed in a matter of seconds. Though effective, problems for these methods exist in the need for an immiscible fluid (oil) that can complicate both the fabrication and use of these techniques on larger scales and the function of ‘dragging’ particles along a surface resulting in a friction-based loss of sample.
By way of a specific example of a microfluidic system of this type, Beebe et al., United States Patent Application Publication No. 2011/0213133, incorporated by reference herein in its entirety, discloses a device and a method for facilitating extraction of a fraction from a biological sample. The biological sample includes non-desired material and a fraction-bound solid phase substrate. The device includes an input zone for receiving the biological sample therein and a second zone for receiving an isolation buffer therein. An output zone receives a reagent therein. A force is movable between a first position adjacent the input zone and a second position adjacent the output zone. The force urges the fraction-bound solid phase substrate from the input zone, through the second zone and into the output zone.
While functional for its intended purpose, the device and method disclosed in the Beebe et al., '133 publication has certain limitations. For example, when the biological sample contains large particulates, debris, precipitates, or other cells that settle out of solution, the efficiency of the recovery and the overall purity of the fraction-bound solid phase substrate decreases as a result of non-desired material impeding the operational path of the fraction-bound solid phase substrate.
In addition, concerning the downstream analysis of the CTCs after isolation, the methods for isolating DNA, RNA, and proteins from complex biological samples are some of the most crucial steps in molecular biology. However, these methods are often overlooked within the biological sample processing workflow. As the throughput of downstream analytical techniques have increased, sample preparation methods have become a limiting factor in overall throughput. Many of the most used methods for sample preparation are very time consuming and can involve many steps including substrate binding, multiple wash steps, dilutions, or other processes that can result in loss of sample or dramatic increases in assay time.
More particularly, when samples of the CTCs of interest are obtained, the current techniques that exist for extraction and purification of RNA and DNA from a single sample are not specifically applicable to analysis of rare cell populations (<1000). This is significantly limiting as biological systems are starting to address these smaller cell populations to understand larger biological processes (e.g. stem cells, CTCs, etc.). The one currently existing assay for circulating tumor cells CTCs is only valid for cell enumeration, without cell purification or nucleic acid extraction/analysis. Other platforms have attempted to capture and analyze CTCs with varying degrees of success. However none can perform protein, DNA and mRNA analysis in an integrated fashion and from a single sample.
Therefore, it is a primary object and feature of the present invention to provide a device and a method isolating a fraction from a biological sample.
It is a further object and feature of the present invention to provide a device and a method for isolating a fraction from a biological sample that is simpler to fabricate, easier to implement and more efficient than prior devices and methods.
It is a still a further object and feature of the present invention to provide a device and a method for isolating a fraction from a biological sample without the significant sample loss associated with prior methods, such as reducing the friction-based losses of the targeted fraction of prior devices/methods.
It is still a further object and feature of the present invention to provide a device that can achieve superior capture of the desired fraction of the biological sample, but that can also perform a comprehensive assay and/or analysis of the fraction using the device without physically contacting the biological sample that encompasses many process for analysis of the fraction including, but not limited to, cell capture, isolation/purification, protein analysis, and DNA and RNA extraction endpoints from a single sample, on as little as a single cell. The device can be used to stain the fraction of the biological sample within the device for imaging analysis, and to extract mRNA and DNA from the fraction without splitting into multiple fractions such that the integrity of the original fraction is maintained and not diluted or washed away, and can therefore be re-sampled for additional analytes. Under this object, the device can be utilized to perform a “fluid biopsy” from a simple blood draw, potentially eliminating the need to perform painful, invasive and expensive tumor biopsies.
In accordance with the present invention, a device is provided for isolating a fraction in a biological sample. The device can take various forms and in one embodiment the fraction is bound to solid phase substrate to define a fraction-bound solid phase substrate. The device includes an input zone for receiving the biological sample therein and an isolation zone for receiving an isolation fluid therein. A force, generally perpendicular to gravity, is movable between a first position adjacent the input zone and a second position adjacent the isolation zone. The force captures the fraction-bound solid phase substrate such that the fraction-hound solid phase substrate moves from the input zone to the isolation zone in response to the force moving from the first position to the second position.
The input zone is partially defined by a lower surface lying in a first plane and wherein the device further comprising a passage having a input communicating with the input zone and an output communicating with the isolation zone. The passage is partially defined by first and second walls. The first and second side walls of the passage at least partially converge from the input to the output thereof. The passage extends along an axis. The axis is vertically spaced from the first plane. The isolation zone is partially defined by a lower surface lying in a second plane, the second plane being between the first plane and the axis. It is contemplated for the force to be a magnetic field. Further, it is contemplated for the force to move from the first position to the second position along a path at least generally transverse to gravity.
In accordance with a further aspect of the present invention, a device is provided for isolating a fraction in a biological sample. The fraction is bound to a solid phase substrate to define a fraction-bound solid phase substrate. The device includes an input zone for receiving the biological sample therein. The input zone is partially defined by a lower surface lying in a first plane. An isolation zone receives an isolation fluid therein. The isolation zone is partially defined by a lower surface lying in a second plane. A passage extends along an axis and has an input communicating with the input zone and an output communicating with the isolation zone. A force captures the fraction-bound solid phase substrate. The force is generally normal to gravity and is movable between a first position adjacent the input zone and a second position adjacent the isolation zone. The captured fraction-bound solid phase substrate moves from the input zone to the isolation zone in response to the force moving from the first position to the second position.
The passage is partially defined by first and second walls. The first and second side walls converge from the input to the output thereof. The axis of the passage is vertically spaced from the first plane and the second plane is between the first plane and the axis. It is contemplated for the force to be a magnetic field. Further, it is contemplated for the force to move from the first position to the second position along a path transverse to gravity and to the force.
In accordance with another aspect of the present invention, the isolation zone can contain a fluid capable of providing an extracellular stain to the fraction bound to the solid phase substrate within the isolation zone or well.
In accordance with a still further aspect of the present invention, a method is provided of isolating a fraction in a biological sample. The method includes the step of providing a biological sample including a fraction-bound solid phase substrate and biological material in an input well. The input well is partially defined by a lower surface lying in a first plane. The fraction-bound solid phase substrate is captured with a force so as to maintain the fraction-bound solid phase substrate at a location above the lower surface of the input well. The biological material is allowed to settle towards the lower surface of the input well and the fraction-bound solid phase substrate is drawn into an isolation well through a passage with the force. The passage extends along an axis vertically spaced above the first plane.
It is contemplated for the force to be generally normal to gravity and to be a magnetic field. The force travels along a path to draw the fraction-bound solid phase substrate from the input well into the isolation well. The path is transverse to gravity. The passage has an input communicating with the input zone and an output communicating with the isolation well or zone. The passage is partially defined by first and second walls. The first and second side walls converge from the input to the output thereof. The isolation well is partially defined by a lower surface lying in a second plane. The second plane is between the first plane and the axis.
In accordance with still another aspect of the present invention, the device also optionally includes a sieve well disposed downstream from the input well, or the isolation well, if present, and joined thereto by a passage having an input communicating with the input or isolation well and an output communicating with the sieve well. The axis of the passage is vertically spaced from the first plane and the passage is partially defined by first and second walls that converge from the input to the output thereof. The sieve well is formed similarly to the isolation well and is partially defined by a lower surface lying in a third plane. The third plane is between the first plane and the axis. The sieve well also includes a separation membrane dividing the sieve well into cavities. The membrane allows for the transfer of fluid between the cavities, while retaining the target or fraction-bound solid phase substrate in the cavity connected to the output of the passage.
In accordance with a still further aspect of the present invention, a method is provided of staining a target in or fraction of a biological sample within the device in order to perform imaging analyses on the fraction within the device. The method includes the step of providing a biological sample including a fraction-bound solid phase substrate and biological material in the input well and moving the fraction using the force from the input well into one of the cavities of the sieve well. The fluid initially present in the sieve well can be used to wash the fraction or target within the sieve well, After washing, the wash fluid is withdrawn out of both cavities of the sieve well via the cavity opposite the cavity holding the fraction. The fluid can pass through the membrane disposed between the cavities in order to be withdrawn from both cavities, while the fraction is retained in the sieve well, such that the solid phase is not physically contacted during the process. A subsequent fluid, such as a fixing and/or permeabilizing fluid, can be introduced into the cavities of the sieve well in a reverse process, allowing the fluid to act upon the fraction, again without physically contacting the fraction. After fixing/permeabilizing the fraction, the fluid can be drawn out of the sieve well and subsequently replaced with a stain in order to effect the fraction and a provide visual indication to a selected component of the fixed/permeabilized fraction. The stain can be removed through the membrane and the fraction can be contacted with a wash fluid in the same manner. The process can be repeated as many times as desired to stain different intracellular components of the fraction to enable the components present in the fraction to be analyzed, such as via a proteomic imaging analysis of the stained fraction without any direct manipulation of the captured cells, minimizing cell damage and loss.
In accordance with still a further object of the present invention, the device also optionally includes a separation well disposed downstream from the input well, or the isolation or sieve well, if present, and joined thereto by a passage having an input communicating with the input well, isolation well or sieve well and an output communicating with the separation well. The axis of the passage is vertically spaced from the first plane and the passage is partially defined by first and second walls that converge from the input to the output thereof. The separation well is formed similarly to the input well and is partially defined by a lower surface lying in a fourth plane. The fourth plane is between the first plane and the axis.
The separation well is connected to a pair of passages each having an input communicating with the separation well and an output communicating with one of a pair cavities formed in an elution well. The axis of each of the passages is vertically spaced from the first plane and the passages are partially defined by first and second walls that converge from the input to the output thereof. The elution well is formed similarly to the separation well and is defined by a lower surface lying in a fifth plane. The fifth plane is between the first plane and the axis.
In accordance with a still further aspect of the present invention, a method is provided of separating the fraction into DNA and RNA fractions for analysis within the device. The fraction-bound solid phase substrate is moved into the separation well from the input well using the force described previously. In the separation well, the fraction-bound solid phase material can be repeatedly interrogated by sequentially adding components with varying chemistries to the separation well to isolate mRNA and DNA from the same sample of the fraction-bound substrate. Following mRNA binding, the force is utilized to draw the mRNA through one of the passages to the corresponding cavity of the elution well, Additionally, DNA is bound by a different component and moved by the force through the other passage to the corresponding separated cavity in the elution well. The samples of the mRNA and the DNA can then be collected from their respective elution well cavities and used for a variety of downstream assays.
In accordance with still another aspect of the present invention, the device can be utilized in a method to perform a fully integrated assay that performs cell capture, purification, protein, genomic and gene expression studies from a single sample on as few as 1-10 cells forming the target or fraction of the biological sample introduced into the device in the method.
In accordance with another embodiment of the present invention, a device is provided for isolating a target or fraction from a biological sample. The target is bound to solid phase substrate to form target bound solid phase substrate. The device includes a lower plate with an upper surface having a plurality of regions. The biological sample is receivable on a first of the regions. An upper plate has a lower surface directed to the upper surface of the lower plate. A force adjacent the upper plate attracts the target bound solid phase substrate toward the lower surface of the upper plate. At least one of the upper plate and the lower plate is movable from a first position wherein the target bound solid phase substrate in the biological sample are drawn to the lower surface of the upper plate and a second position wherein the target bound solid phase substrate are isolated from the biological sample.
The regions of the lower plate are hydrophilic and the portions of the upper surface of the outside of the regions of the lower plate are hydrophobic. The lower surface of the upper plate is also hydrophobic. The upper plate is axially movable between the first and second positions or is rotatably between the first and second positions. The upper surface of the lower plate and lower surface of the upper surface are spaced by a predetermined distance.
In accordance with a further aspect of the present invention, a method is provided for isolating a target from a biological sample. The target is bound to solid phase substrate to form target bound solid phase substrate. The method includes the steps of providing the biological sample at a region of a surface of a lower plate and positioning an upper plate in spaced relation to the lower plate. The upper plate has a lower surface directed to the upper surface of the lower plate. The target bound solid phase substrate is drawn toward the lower surface of the upper plate with a force. At least one of the lower plate and the upper plate is moved from a first position wherein the target bound solid phase substrate in the biological sample are drawn toward the lower surface of the upper plate to a second position wherein the target bound solid phase substrate are isolated from the biological sample.
The upper surface of the lower plate may include a plurality of regions that are hydrophilic. The upper surface of the lower surface outside of the regions is hydrophobic. The lower surface of the upper plate is hydrophobic. The upper plate moves along a longitudinal axis between the first and second positions or is rotatable between the first and second positions. It is contemplated to space the upper surface of the lower plate and lower surface of the upper surface by a predetermined distance.
In accordance with a still further aspect of the present invention, a method is provided for isolating a target from a biological sample. The target is bound to solid phase substrate to form target bound solid phase substrate. The method includes the step of providing the biological sample at a first region of a surface of a first plate. A fluid is deposited on a second region of the surface of the first plate. A second plate is positioned in spaced relation to the first plate. The second plate has a hydrophobic surface directed towards the surface of the first plate. The target bound solid phase substrate is drawn toward the surface of the second plate with a force. At least one of the first plate and the second plate is moved from a first position wherein the target bound solid phase substrate in the biological sample are drawn toward the surface of the second plate to a second position wherein the target bound solid phase substrate are isolated from the biological sample.
The portions of the surface of the first plate outside of the first and second regions are hydrophobic. The second plate moves along a longitudinal axis between the first and second positions or is rotatable between the first and second positions. The surface of the second plate is spaced from the surface of the first plate by a predetermined distance. It is intended for the force to be magnetic and for the target bound solid phase substrate to be received in the fluid with the at least one of the first plate and the second plate in the second position. The fluid can be selected to be able to be utilized to wash, fix, permeabilize, stain or extract RNA or DNA from the target substance, or in any combination thereof. The method may also include the step of isolating the target bound solid phase substrate from the force.